Proteomics profiling of chimeric-truncated tissue plasminogen activator producing- Chinese hamster ovary cells cultivated in a chemically defined medium supplemented with protein hydrolysates

Background: Culture media enrichment through the addition of protein hydrolysates is beneficial for achieving higher protein expression

Methods: In this study, designing the optimum mixture of four soy and casein-derived hydrolysates was successfully performed by design of experiment and specific productivity increased in all predicted combinations. Protein profile of recombinant CHO [rCHO] cells producing tissue plasminogen activator in a serum-free medium [SFM] supplemented with designed hydrolysate additives was compared to that of rCHO cells cultivated in SFM

Results: Identification of differentially expressed proteins using two-dimensional gel electrophoresis coupled with MALDI-TOF/TOF revealed the role of energy metabolism related proteins and importance of prevention of oxidative stress by this special media enrichment strategy. Up-regulation of mitochondrial enzymes, pyruvate dehydrogenase E1 and Peroxiredoxin-III, as well as other proteins involved in metabolic pathways, and uridine monophosphate/cytidine monophosphate kinase indicated higher metabolic activity. Furthermore, along with antioxidant effect of peptones, proteins with antioxidant function such as ferritin and peroxiredoxin-III were up-regulated

Conclusion: Understanding molecular mechanisms involved in enhancement of protein expression can provide new approaches for efficiently engineering rCHO cell. These results support the competence of proteomics studies in finding new insights to biochemical pathways for a knowledge-based optimization of media compositions

Utilization of site-specific recombination in biopharmaceutical production

Mammalian expression systems, due to their capacity in post-translational modification, are preferred systems for biopharmaceutical protein production. Several recombinant protein systems have been introduced to the market, most of which are under clinical development. In spite of significant improvements such as cell line engineering, introducing novel expression methods, gene silencing and process development, expression level is unpredictable and unstable because of the random location of integration in the genome. Site-specific recombination techniques are capable of producing stable and high producer clonal cells; therefore, they are gaining more importance in the biopharmaceutical production. Site-specific recombination methods increase the recombinant protein production by specifically inserting a vector at a locus with specific expression trait. The present review focused on the latest developments in site-specific recombination techniques, their specific features and comparisons

Effects of peptone supplementation in different culture media on growth, metabolic pathway and productivity of CHO DG44 cells; a new insight into amino acid profiles

The optimization of bioprocess conditions towards improved growth profile and productivity yield is considered of great importance in biopharmaceutical manufacturing. Peptones as efficient sources of nutrients have been studied for their effect on media development; however, their role on metabolic pathway is not well understood. In the present study, the effect of different concentration of peptones on a recombinant Chinese hamster ovary [CHO] cell line grown in three serum-free suspension cultures was determined. Six peptones of different origins and available amino acid profiles were investigated regarding their impact on cell growth, productivity, and metabolic pathways changes. In optimized feeding strategies, increases of 136% and 159% in volumetric productivity [for a low-nutrient culture media] and 55% [for a high-nutrient culture media] were achieved. Furthermore, particular sources of peptones with specific amino acid profile developed preferential results for each different culture medium. Two peptones, SoyA2SC and SoyE-110, were the only hydrolysates that showed production improvement in all three media. Casein Peptone plus Tryptone N1 and SoyA3SC showed different improved results based on their implemented concentration for each individual basal medium. The amino acid profile of peptones may provide clues to identify the most effective feeding strategies for recombinant CHO cells

Effect of peptone feeding on transient gene expression process in CHO DG44

Transient Gene Expression [TGE] gained popularity over the last decade as a rapid method for the production of milligram to gram quantities of recombinant proteins for preclinical studies in biophama industry. Thereby, the optimization of the TGE technique for Chinese hamster ovary [CHO] as the dominant host for the production of bio therapeutics is of great interest to reach the values for Human Embryo Kidney-293 [HEK-293] cells in terms of transfection efficiencies and production titers. TGE efficiencies are cell line and vector dependent. In transfection efficiency optimization experiments, different starting cell densities, different amounts of plasmid DNA and PEI transfection reagent were investigated to achieve the best conditions leading to maximum transfection efficiencies. Furthermore, in order to investigate the effect of peptone feeding on transfection efficiency, three different sources of peptones with the greatest effect in the CD DG44 basal media were selected; Casein Tryptone N1, Soy petone A2SC and Soy peptone E110. The transfection strategy performed here was able to make an outstanding increase in transfection efficiency of CHO DG44 cell line transfected with pTracer-SV40-mutated t-PA plasmid from 3.6% in our starting nonoptimized condition to 66.93% in finally optimized situation. Moreover, peptone feeding strategy used here was successful to increase volumetric productivities up to 37%. In addition, the amounts of both PEI and plasmid DNA were reduced up to 66% and 25%, respectively compared to our previous protocol. Here we described an optimization process for TGE in suspensionadapted CHO cells based on Polyethylenimine [PEI]/DNA concentration, DNA: PEI ratio, starting cell densities and peptone feeding strategy

Periplasmic expression of a novel human bone morphogenetic protein-7 mutant in escherichia coli

Bone Morphogenetic Proteins [BMPs] belong to the transforming growth factor-beta [TGF-beta] superfamily, and play an important role in bone metabolism. Recombinant forms of BMP-2 and BMP-7 are the only BMPs used clinically. In this study the mature part of human bone morphogenetic protein-7 [BMP-7] was engineered through substitution of the BMP-7 Nterminal sequence by heparin-binding site of BMP-2. This targeted substitution was made to enhance the binding affinity of the novel protein to the extracellular matrix components such as heparin and heparan sulfate proteoglycans [HSPGs]. The engineered protein was expressed in Escherichia coli [E.coli]. The PelB signal sequence was used to translocate soluble proteins into the periplasmic space of E.coli. The protein was purified from periplasmic extract using Ni-NTA chromatography and the SDS-PAGE and western blot analysis confirmed the successful expression of the novel protein. The novel hBMP-7 mutant was produced as approximately 16 kDa monomer. It was found that the heparin binding of this protein was approximately 50% more than that of the wild-type at a protein concentration of 500 ng/ml. The findings showed that the periplasmic expression may be suitable to produce complex proteins like BMPs

Human tissue plasminogen activator expression in Escherichia coli using cytoplasmic and periplasmic cumulative power

Tissue plasminogen activator [tPA] is a serine protease, which is composed of five distinct structural domains with 17 disulfide bonds, representing a model of high-disulfide proteins in human body. One of the most important limitations for high yield heterologous protein production in Escherichia coli [E. coli] is the expression of complex proteins with multiple disulfide bridges. In this study the combination of two distinct strategies, manipulated cytoplasm and native periplasm, was applied to produce the functional full length tPA enzyme in E. coli. Using a PelB signal peptide sequence at 5' site of tPA gene, the expression cassette was prepared and subsequently was transformed into a strain with manipulated oxidizing cytoplasm. Then the induction was made to express the protein of interest. The SDS-PAGE analysis and gelatin hydrolysis confirmed the successful expression of functional tPA. The results of this study showed that complex proteins can be produced in E. coli using the cumulative power of both cytoplasm and periplasm

Simultaneous spectrophotometric determination of mixtures of food colorants

Mixtures of food colorants, containing new coccine, ponceaue 6R and scarlet GN were simultaneously analyzed by spectrophotometry without previous chemical separation. Sixty mixtures of colorants with three-components were evaluated and the spectrograms were smoothed through the use of seven experimental points. The multivariate data consisted of normal, first- and second-derivative absorbance spectra [delta lambda=5 nm] registered from 300-650 nm. The data obtained from experiments were processed by PLS method and the proposed method was applied satisfactorily for determination of these colorants in two commercial food products